Single-chain Fv (sFv) proteins are genetically engineered molecules that consist of the two variable domains of an antibody or T cell receptor connected by a polypeptide linker and that contain the antigen binding function of the parental protein in a single 30 kD polypeptide chain. (Huston, J. S., et al., Proc. Natl. Acad. Sci. U.S.A. 85:5879-5883 (1988); Bird, R. E., et al. Science 242:423-426 (1988); Huston, J. S., et al., Meth. Enzymol. 203:46-88 (1991)).
The Fv portion of an antibody is the smallest fragment to bear the complete antigen-binding site. It is a 25 kD heterodimer consisting of the N-terminal variable (V) domains of the heavy (H) and light (L) chain. (Inbar, D., et al., Proc. Natl. Acad. Sci. U.S.A. 69:2659-2662 (1972); Hochman, J., Biochemistry 15:2706-2710 (1976); Hochman, J., et al., Biochemistry 12:1130-1135 (1973)). More recently, a genetically engineered single-chain Fv (sFv) with antigen binding activity has been produced by connecting the C-terminus of one V domain to the N-terminus of the other with a peptide linker. Huston, J. S., et al., Proc. Natl. Acad. Sci U.S.A. 85:5879-5883 (1988); and Bird, R. E., et al., Science 242:423-426 (1988) Since then, sFv proteins have been produced from a large number of different antibodies (Huston, J. S., et al., Intern. Rev. Immunol. 10:195-217 (1993); Winter, G. and Milstein, C. Nature 349:293-299 (1991)) and initial studies (Kurucz, I., et al., Proc. Natl. Acad. Sci. U.S.A. 90:3830-3834 (1993); Novotny, J., et al., Proc. Natl. Acad. Sci. USA 88:8646-8650 (1991); Soo Hoo, W. F., et al., Proc. Natl. Acad. Sci. USA 89:4759-4763 (1992); Ward, E. S., J. Mol. Biol. 224:885-890 (1992)) have described the production of sFv analogues of T cell receptors (TcR), cell surface molecules that are highly homologous to immunoglobulins (Hedrick, S. M., et al., Nature 308:153-158 (1984); Davis, M. M. and Bjorkman, P. J., Nature 334, 395-402 (1988)).
Most sFv proteins have been generated in bacteria, often as insoluble, cytoplasmic inclusion bodies. Protein from inclusion bodies is not active and must be solubilized, renatured in vitro and oxidized to form parent disulfide bonds, (Huston, J. S., et al. Methods Enzymol. 203:46-78 (1991)). Alternatively the introduction of an N-terminal leader sequence can direct sFv into the periplasmic space of bacteria by a secretion process wherein the leader sequence is removed (Holland, I. B., et al., Methods Enzymol. 182:132-143 (1990)) and protein folding is accomplished, aided by enzymes that catalyze disulfide bond formation (Bardwell, J. C. A., et al., Cell 67:581-589 (1991)) and cis-trans isomerization of proline residues (Hayano, T., et al., Biochemistry 30:3041-3048 (1991)). However, even with these enzymes, secreted sFv proteins sometimes exist as insoluble aggregates in the periplasmic space, which must be solubilized and refolded in vitro (Johnson, S. and Bird, R. E., Methods Enzymol. 203:88-98 (1991); George, A. J. T., et al., J. Immunol. 152, in press (1994)). Knappik, A., et al. (Bio/Technology 11:77-83 (1993)) have recently attempted to overcome this problem by overexpressing protein disulfide isomerase and prolyl cis-trans isomerase in the piroplasm of bacteria. However, neither enzyme induced a significant change in folding efficiency of sFv proteins when expressed either alone or together with the other enzyme.
A different approach to produce active sFv would be to use the more sophisticated refolding machinery that is located in the endoplasmic reticulum (ER) of mammalian cells. The potential benefit of this approach could be substantial, since the ER not only contains enzymes that catalyze specific isomerization steps but it also contains a number of proteins (e.g., chaperones) that aid in the folding process and prevent the secretion of incorrectly folded proteins (Gething, M. J. and Sambrook, J. Nature 355:33-45 (1992); Pelham, H. R., Annu. Rev. Cell Biol. 5:1-23 (1989); Hurtley, S. M. and Helenius, A., Annu. Rev. Cell Biol. 5:277-307 (1989)). A number of sFv fusion proteins have been expressed in or on the surface of mammalian cells. Examples include an anti-HIV-KDEL (SEQ ID NO: 18) fusion protein, or anti-HIV sFv alone, that remains bound in the ER (Marasco, W. A., et al. Proc. Natl. Acad. Sci. USA 90:7889-7893 (1993)) and anti-tumor sFv proteins fused to TcR-.zeta. or Fc.sub..epsilon. RI-.gamma. that trigger cell mediated cytolysis (Eshhar, Z., et al. Proc. Natl. Acad. Sci. USA 90:720-724 (1993); Hwu, P., et al. J. ExP. Med. 178:361-366 (1993); Stancovski, I., et al. J. Immunol. 151:6577-6582 (1993)). However, production of this class of proteins by mammalian cells is generally very low, varying from a few micrograms to a few milligrams, if production is possible at all. (Davis, S. J., et al., J. Biol. Chem. 265:10410-10418 (1990); Traaunecker, A. et al., EMBO J., 10:3655-3659 (1991)).
To date, it is not certain what the rate-limiting step, or steps, in the efficient expression and secretion of sFv proteins in mammalian cells may be, nor has it been apparent how to induce, or increase existing production levels.